The goal is to elucidate the structures functional roles and physiological regulation of the expression of multiple forms of type II cAMP-dependent protein kinases (PKII). We have shown that the basis for PKII diversity is the tissue-specific expression of several distinct cAMP-binding or regulatory (RII) subunits. Cloned cDNA sequences corresponding to mRNA for a major cerebral cortex RII isoform (RII-B) will be isolated from a Lambda gt11 library. Th RII-B cDNA probe will be used to obtain cDNA clones for RII subunit isoforms produced in non-neural bovine tissues (RII-H) and murine erythroleukemic cells (RII-52 and RII-54) from Lamba gt11 or pU C18 cDNA libraries. The number and sizes of mRNAs coding for RII subunits in adult tissues will be determined. Northern gels and dot-blot analyses will be used to quantify changes in RII mRNA content during the induction of RII-52 by cAMP treatment and erythroid differentiation. The possibility that regulation of RII-52 biosynthesis is exerted at the levels of transcription, processing and/or mRNA stabilization will be examined. The degree to which the RII isoforms are homologous and divergent will be investigated by RNAse mapping and primer extension analysis, and subsequently, direct sequencing using the Sanger chain termination procedure. cDNA probes will be used to isolate cloned genomic sequences for the RII isoforms. THe organization of RII genes will be established by restriction and S1 mapping and sequencing 1 kb upstream from the 5' end of the structural gene to begin to assess the DNA sequences involved in the differential tissue-specific, developmental and hormone stimulated expression of RII isoforms. Finally, P75, a calmodulin binding protein that complexes RII-B, will be purified to homogeneity and characterized by hydrodynamic methods. Binding and interactions among P75, calmodulin and RII-B will be quantitatively analyzed and effects of phospho/dephospho-P75 on the functional properties of PKII-B will be determined.